Methods and compositions for herbicide tolerance in plants

ABSTRACT

The invention relates to biotechnology and provides novel recombinant DNA molecules and engineered proteins for conferring tolerance to protoporphyrinogen oxidase-inhibitor herbicides. The invention also provides herbicide tolerant transgenic plants, seeds, cells, and plant parts containing the recombinant DNA molecules, as well as methods of using the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority of U.S. ProvisionalApplication No. 62/200,428, filed Aug. 3, 2015, the disclosure of whichis hereby incorporated by reference in its entirety.

INCORPORATION OF SEQUENCE LISTING

The sequence listing that is contained in the file namedMONS383US_ST25.txt, which is 71,195 bytes (measured in MS-WINDOWS) andcreated on Jun. 27, 2016, is filed herewith by electronic submission andincorporated herein by reference.

BACKGROUND

Field of the Invention

The present invention relates to the field of biotechnology. Morespecifically, the invention relates to recombinant DNA moleculesencoding enzymes that provide tolerance to herbicides that inhibitprotoporphyrinogen oxidase.

Related Art

Agricultural crop production often utilizes transgenic traits createdusing the methods of biotechnology. A heterologous gene, also known as atransgene, can be introduced into a plant to produce a transgenic trait.Expression of the transgene in the plant confers a trait, such asherbicide tolerance, on the plant. Examples of transgenic herbicidetolerance traits include glyphosate tolerance, glufosinate tolerance,and dicamba tolerance. With the increase of weed species resistant tothe commonly used herbicides, new herbicide tolerance traits are neededin the field. Herbicides of particular interest include herbicides thatinhibit protoporphyrinogen oxidase (PPO), referred to as PPO herbicides.PPO herbicides provide control of a spectrum of herbicide-resistantweeds, thus making a trait conferring tolerance to these herbicidesparticularly useful in a cropping system combined with one or more otherherbicide-tolerance trait(s).

Protoporphyrinogen oxidase functions in both chlorophyll and hemebiosynthesis pathways where it converts protoporphyrinogen IX toprotoporphyrin IX. Following production of protoporphyrin IX, thechlorophyll and heme biosynthetic pathways diverge with different metalions being incorporated (iron for heme and magnesium for chlorophyll).Segments of this pathway are conserved across prokaryotes andeukaryotes, and many of the PPO enzymes found across prokaryotes andeukaryotes are relatively similar. Some prokaryotes (e.g.,cyanobacteria) use this pathway for chlorophyll and heme productionwhile other prokaryotes (e.g., Escherichia coli) use this pathway forheme production.

Herbicide-insensitive protoporphyrinogen oxidases (“iPPOs”) have beenisolated from a number of prokaryotes and eukaryotes. On a structuralbasis, it is believed that there are at least three distinct subclassesof PPO enzymes: HemY (Hans son and Hederstedt, “Cloning andcharacterization of the Bacillus subtilis hemEHY gene cluster, whichencodes protoheme IX biosynthetic enzymes” Journal of Bacteriology174(24):8081-8093 (1992)), HemG (Sasarman, et al., “Mapping of a new hemgene in Escherichia coli K12” Microbiology 113:297-303 (1979)), and HemJ(Boynton, et al., “Discovery of a gene involved in a third bacterialprotoporphyrinogen oxidase activity through comparative genomic analysisand functional complementation” Applied and Environmental Microbiology77(14):4795-4801 (2011)). This invention provides novel recombinantiPPOs that are members of the HemG family. Despite twenty years ofresearch and the number of iPPOs identified to date, a transgenic cropplant comprising a recombinant iPPO has yet to be commercialized. Astrong weed control platform depends, in part, on continued developmentof herbicide tolerance trait packages. Identifying and utilizing iPPOsto create transgenic crop traits therefore represents an advance toagriculture.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a recombinant DNA moleculecomprising a heterologous promoter operably linked to a nucleic acidsequence encoding a polypeptide that has at least 85% sequence identityto a polypeptide sequence chosen from SEQ ID NOs:1-20, wherein thepolypeptide has herbicide-insensitive protoporphyrinogen oxidaseactivity. In certain embodiments, the polypeptide has at least about 85%sequence identity, at least about 90% sequence identity, at least 95%sequence identity, at least 96% sequence identity, at least 97% sequenceidentity, at least 98% sequence identity, or at least 99% sequenceidentity to a polypeptide sequence chosen from among SEQ ID NOs:1-20 andhas herbicide-insensitive protoporphyrinogen oxidase activity. In someembodiments there is provided a recombinant DNA molecule, wherein thenucleic acid sequence is selected from the group consisting of SEQ IDNOs:22-63. In particular embodiments the recombinant DNA moleculeencodes a polypeptide comprising an amino acid sequence selected fromthe group consisting of SEQ ID NOs:1-20. A recombinant polypeptide thatcomprises at least 85% sequence identity to the full length of an aminoacid sequence chosen from among SEQ ID NOs:1-20, wherein the polypeptidehas herbicide-insensitive protoporphyrinogen oxidase activity istherefore provided by the invention.

In certain embodiments a heterologous promoter, for instance, a promoterfunctional in a plant cell, is operably linked to the nucleic acidsequence encoding a polypeptide that has at least 85% sequence identityto a polypeptide sequence of the invention, for instance a polypeptidesequence chosen from SEQ ID NOs:1-20, wherein the polypeptide hasherbicide-insensitive protoporphyrinogen oxidase activity. Such aresulting DNA molecule may further comprise a targeting sequence thatfunctions to localize the polypeptide within a cell.

In one aspect, the invention provides a DNA construct comprising arecombinant DNA molecule of the invention. In one embodiment, such a DNAconstruct comprises, in operable linkage to a nucleic acid sequence ofthe invention, a targeting sequence that functions to localize thepolypeptide within a cell. The DNA molecule may be present in the genomeof a transgenic plant, seed, or cell. In certain embodiments, thepolypeptide confers herbicide tolerance to the cell, plant, seed, orplant part.

Another aspect of the invention provides a transgenic plant, seed, cell,or plant part comprising a recombinant DNA molecule of the invention ora recombinant polypeptide of the invention. The transgenic plant, seed,cell, or plant part may thus comprise, i.e. display, tolerance to atleast one PPO herbicide. In some embodiments the transgenic plant, seed,cell, or plant part comprises an additional transgenic herbicidetolerance trait.

Another aspect of the invention provides a method for conferringherbicide tolerance to a plant, seed, cell, or plant part comprising:heterologously expressing a recombinant polypeptide of the invention inthe plant, seed, cell, or plant part. In some embodiments of the method,the plant, seed, cell, or plant part comprises protoporphyrinogenoxidase activity conferred by the recombinant polypeptide. In someembodiments the herbicide tolerance is to at least one PPO herbicideselected from the group consisting of acifluorfen, fomesafen, lactofen,fluoroglycofen-ethyl, oxyfluorfen, flumioxazin, azafenidin,carfentrazone-ethyl, sulfentrazone, fluthiacet-methyl, oxadiargyl,oxadiazon, pyraflufen-ethyl, saflufenacil and S-3100.

Another aspect of the invention relates to a method of planttransformation, comprising the steps of: a) introducing a recombinantDNA molecule of the invention into a plant cell; and b) regenerating atransgenic plant therefrom that comprises the recombinant DNA molecule.The method may further comprise the step of selecting a plant that istolerant to at least one PPO herbicide. The method may also furthercomprise a step of crossing the regenerated plant with itself or with asecond plant and collecting seed from the cross.

Yet another aspect of the invention provides a method for controllingweeds in a plant growth area, comprising contacting a plant growth areacomprising the transgenic plant or seed with at least one PPO herbicide,wherein the transgenic plant or seed is tolerant to the PPO herbicideand wherein weeds are controlled in the plant growth area.

Also provided is a method of identifying a nucleotide sequence encodinga protein having protoporphyrinogen oxidase activity, the methodcomprising: a) transforming an E. coli strain having a gene knockout forthe native E. coli PPO enzyme with a bacterial expression vectorcomprising a recombinant DNA molecule encoding a candidate herbicidetolerance protein; and b) growing said transformed E. coli using aheme-free bacterial medium, wherein growth using said bacterial mediumidentifies a protein having protoporphyrinogen oxidase activity.

Further provided by the invention is a method of identifying anucleotide sequence encoding a protein having herbicide-insensitiveprotoporphyrinogen oxidase activity, the method comprising: a)transforming an E. coli strain having a gene knockout for the native E.coli PPO enzyme with a bacterial expression vector comprising arecombinant DNA molecule encoding a recombinant protein; and b) growingsaid transformed E. coli using a bacterial medium containing at leastone PPO herbicide, wherein growth of bacteria identifies a proteinhaving herbicide-insensitive protoporphyrinogen oxidase activity.

Another aspect of the invention relates to a method of screening for aherbicide tolerance gene comprising: a) expressing a recombinant DNAmolecule of the invention in a plant cell; and b) identifying a plantcell that displays tolerance to a PPO herbicide.

Further, the invention provides methods of screening for a herbicidetolerance gene comprising: a) expressing a recombinant DNA molecule ofthe invention in a bacterial cell lacking HemG, wherein the bacterialcell is grown in a heme-free medium in the presence of a PPO herbicide;and b) identifying a bacterial cell that displays tolerance to a PPOherbicide.

In another aspect, the invention provides a method of producing a planttolerant to a PPO herbicide and at least one other herbicide comprising:a) obtaining a plant comprising a recombinant DNA molecule of theinvention; b) crossing the transgenic plant with a second plantcomprising tolerance to the at least one other herbicide, and c)selecting a progeny plant resulting from said crossing that comprisestolerance to a PPO herbicide and the at least one other herbicide isanother aspect of the invention.

The invention also provides, in another aspect, a method for reducingthe development of herbicide tolerant weeds comprising: a) cultivatingin a crop growing environment a plant of the present invention thatcomprises tolerance to a PPO herbicide, for instance by comprising a DNAmolecule of the present invention, and comprises tolerance to at leastone other herbicide; and b) applying a PPO herbicide and at least oneother herbicide to the crop growing environment, wherein the crop plantis tolerant to the PPO herbicide and the at least one other herbicide.In certain embodiments of the method, the PPO herbicide may be selectedfrom the group consisting of acifluorfen, fomesafen, lactofen,fluoroglycofen-ethyl, oxyfluorfen, flumioxazin, azafenidin,carfentrazone-ethyl, sulfentrazone, fluthiacet-methyl, oxadiargyl,oxadiazon, pyraflufen-ethyl, saflufenacil and S-3100. In someembodiments of the method, the at least one other herbicide is selectedfrom the group consisting of: an ACCase inhibitor, an ALS inhibitor, anEPSPS inhibitor, a synthetic auxin, a photosynthesis inhibitor, aglutamine synthesis inhibitor, a HPPD inhibitor, a PPO inhibitor, and along-chain fatty acid inhibitor. In particular embodiments, the ACCaseinhibitor is an aryloxyphenoxy propionate or a cyclohexanedione; the ALSinhibitor is a sulfonylurea, imidazolinone, triazoloyrimidine, or atriazolinone; the EPSPS inhibitor is glyphosate; the synthetic auxin isa phenoxy herbicide, a benzoic acid, a carboxylic acid, or asemicarbazone; the photosynthesis inhibitor is a triazine, a triazinone,a nitrile, a benzothiadiazole, or a urea; the glutamine synthesisinhibitor is glufosinate; the HPPD inhibitor is an isoxazole, apyrazolone, or a triketone; the PPO inhibitor is a diphenylether, aN-phenylphthalimide, an aryl triazinone, or a pyrimidinedione; or thelong-chain fatty acid inhibitor is a chloroacetamide, an oxyacetamide,or a pyrazole.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Alignment of H_N90, H_N20, H_N60, H_N10, H_N30, H_N40, H_N50,H_N70, H_N100, and H_N110 protein sequences (SEQ ID NOs:1-10), withconsensus positions shown below.

FIG. 2. Assay results from PPO bacterial screening system with PPOherbicides measured at 8 hours of growth of E. coli containing thetested iPPO.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is the amino acid sequence of H_N90.

SEQ ID NO:2 is the amino acid sequence of H_N20.

SEQ ID NO:3 is the amino acid sequence of H_N60.

SEQ ID NO:4 is the amino acid sequence of H_N10, which is the E. coliwild-type HemG protoporphyrinogen oxidase (NCBI GenBank Accession No.WP_21498199).

SEQ ID NO:5 is the amino acid sequence of H_N30.

SEQ ID NO:6 is the amino acid sequence of H_N40.

SEQ ID NO:7 is the amino acid sequence of H_N50.

SEQ ID NO:8 is the amino acid sequence of H_N70.

SEQ ID NO:9 is the amino acid sequence of H_N100.

SEQ ID NO:10 is the amino acid sequence of H_N110.

SEQ ID NO:11 through SEQ ID NO:17 are amino acid sequences lacking thestart methonine corresponding to SEQ ID NO:1, 2, 4, 5, 6, 7, and 9,respectively.

SEQ ID NO:18 and SEQ ID NO:19 are amino acid variants of SEQ ID NO:11.

SEQ ID NO:20 is an amino acid variant of SEQ ID NO:17.

SEQ ID NO:21 is the amino acid sequence of the WH, which is thewild-type protoporphyrinogen oxidase from Amaranthus tuberculatus(waterhemp).

SEQ ID NO:22 through SEQ ID NO:31 are nucleotide sequences encoding SEQID NO.: 1 through SEQ ID NO:10, respectively, codon optimized for E.coli expression.

SEQ ID NO:32 through SEQ ID NO:41 are the nucleotide sequences encodingSEQ ID NO: 1 through SEQ ID NO:10, respectively, codon optimized fordicot expression.

SEQ ID NO:42 through SEQ ID NO:48 are the nucleotide sequences encodingSEQ ID NO:11 through SEQ ID NO:17, respectively, codon optimized fordicot expression.

SEQ ID NO:49 and SEQ ID NO:52 are nucleotide variants of SEQ ID NO:11and SEQ ID NO:12, respectively.

SEQ ID NOs:50, 51 and 53 are nucleotide sequences encoding SEQ IDNOs:18, 19, and 20.

SEQ ID NO:54 through SEQ ID NO:63 are the nucleotide sequences encodingSEQ ID NO: 1 through SEQ ID NO:10, respectively, codon optimized formonocot expression.

DETAILED DESCRIPTION

The following descriptions and definitions are provided to better definethe invention and to guide those of ordinary skill in the art in thepractice of the invention. Unless otherwise noted, terms are to beunderstood according to conventional usage by those of ordinary skill inthe relevant art.

The invention provides novel, recombinant DNA molecules and proteinsthat encode herbicide-insensitive protoporphyrinogen oxidases (iPPOs).For instance, the invention provides in one embodiment vectors andexpression cassettes encoding microbially derived iPPOs for expressionin cells and plants. Methods for producing cells and plants tolerant toPPO herbicides are also provided. The invention further provides methodsand compositions for using protein engineering and bioinformatic toolsto obtain and improve iPPOs.

In specific aspects, the invention provides recombinant DNA moleculesand proteins. As used herein, the term “recombinant” refers to anon-naturally occurring DNA, protein, cell, seed, or organism that isthe result of genetic engineering and as such would not normally befound in nature. A “recombinant DNA molecule” is a DNA moleculecomprising a DNA sequence that does not naturally occur in nature and assuch is the result of human intervention, such as a DNA moleculecomprised of at least two DNA molecules heterologous to each other. Anexample of a recombinant DNA molecule is a DNA molecule provided hereinencoding herbicide-insensitive protoporphyrinogen oxidase operablylinked to a heterologous regulatory or other element, such as aheterologous promoter. A “recombinant protein” is a protein comprisingan amino acid sequence that does not naturally occur and as such is theresult of human intervention, such as an engineered protein or achimeric protein. A recombinant cell, seed, or organism is a cell, seed,or organism comprising transgenic DNA, for example a transgenic cell,seed, plant, or plant part comprising a recombinant DNA molecule andtherefore produced as a result of plant transformation.

As used herein, the term “genetic engineering” refers to the creation ofa non-natural DNA, protein, or organism that would not normally be foundin nature and therefore entails applying human intervention. Geneticengineering can be used to produce an engineered DNA, protein, ororganism that was conceived of and created in the laboratory using oneor more of the techniques of biotechnology such as molecular biology,protein biochemistry, bacterial transformation, and planttransformation. For example, genetic engineering can be used to create achimeric gene comprising at least two DNA molecules heterologous to eachother using one or more of the techniques of molecular biology, such asgene cloning, DNA ligation, and DNA synthesis. A chimeric gene mayconsist of two or more heterologous DNA molecules that are operablylinked, such as a protein-coding sequence operably linked to a geneexpression element such as a transit peptide-coding sequence or aheterologous promoter. Genetic engineering can be used to create anengineered protein whose polypeptide sequence was created using one ormore of the techniques of protein engineering, such as protein designusing site-directed mutagenesis and directed evolution using randommutagenesis and DNA shuffling. An engineered protein may have one ormore deletions, insertions, or substitutions relative to the codingsequence of the wild-type protein and each deletion, insertion, orsubstitution may consist of one or more amino acids. In anotherembodiment, an engineered protein may consist of two heterologouspeptides that are operably linked, such as an enzyme operably linked toa transit peptide.

As used herein, “herbicide-insensitive” means the ability of aprotoporphyrinogen oxidase (PPO) to maintain at least some of itsenzymatic activity in the presence of one or more PPO herbicide(s).Enzymatic activity of a protoporphyrinogen oxidase can be measured byany means known in the art, for example, by an enzymatic assay in whichthe production of the product of protoporphyrinogen oxidase or theconsumption of the substrate of protoporphyrinogen oxidase in thepresence of one or more PPO herbicide(s) is measured via fluorescence,high performance liquid chromatography (HPLC), or mass spectrometry(MS). Another example of an assay for measuring enzymatic activity of aprotoporphyrinogen oxidase is a bacterial assay, such as the growthassays described herein, whereby a recombinant protoporphyrinogenoxidase is expressed in a bacterial cell otherwise lacking PPO activityand the ability of the recombinant protoporphyrinogen oxidase tocomplement this knockout phenotype is measured. Herbicide-insensitivitymay be complete or partial insensitivity to a particular herbicide, andmay be expressed as a percent (%) tolerance or insensitivity to aparticular PPO herbicide. As used herein, an “herbicide-insensitiveprotoporphyrinogen oxidase” or “iPPO” exhibits herbicide-insensitivityin the presence of one or more PPO herbicide(s).

As used herein, a “hemG knockout strain” means an organism or cell of anorganism, such as E. coli, that lacks HemG activity to the extent thatit is unable to grow on heme-free growth medium, or such that its growthis detectably impaired in the absence of heme relative to an otherwiseisogenic strain comprising a functional HemG. A hemG knockout strain of,for instance, E. coli may be prepared in view of knowledge in the art,for instance in view of the E. coli hemG sequence (Ecogene Accession No.EG11485; Sasarman et al., “Nucleotide sequence of the hemG gene involvedin the protoporphyrinogen oxidase activity of Escherichia coli K12” CanJ Microbiol 39:1155-1161, 1993).

As used herein, the term “transgene” refers to a DNA moleculeartificially incorporated into an organism's genome because of humanintervention, such as a plant transformation method. As used herein, theterm “transgenic” means comprising a transgene, for example a“transgenic plant” refers to a plant comprising a transgene in itsgenome and a “transgenic trait” refers to a characteristic or phenotypeconveyed or conferred by the presence of a transgene incorporated intothe plant genome. Because of such genomic alteration, the transgenicplant is something distinctly different from the related wild-type plantand the transgenic trait is a trait not naturally found in the wild-typeplant. Transgenic plants of the invention comprise the recombinant DNAmolecules and engineered proteins provided by the invention.

As used herein, the term “heterologous” refers to the relationshipbetween two or more items derived from different sources and thus notnormally associated in nature. For example, a protein-coding recombinantDNA molecule is heterologous with respect to an operably linked promoterif such a combination is not normally found in nature. In addition, aparticular recombinant DNA molecule may be heterologous with respect toa cell, seed, or organism into which it is inserted when it would notnaturally occur in that particular cell, seed, or organism.

As used herein, the term “isolated” refers to at least partiallyseparating a molecule from other molecules typically associated with itin its natural state. In one embodiment, the term “isolated” refers to aDNA molecule that is separated from the nucleic acids that normallyflank the DNA molecule in its natural state. For example, a DNA moleculeencoding a protein that is naturally present in a bacterium would be anisolated DNA molecule if it was not within the DNA of the bacterium fromwhich the DNA molecule encoding the protein is naturally found. Thus, aDNA molecule fused to or operably linked to one or more other DNAmolecule(s) with which it would not be associated in nature, for exampleas the result of recombinant DNA or plant transformation techniques, isconsidered isolated herein. Such molecules are considered isolated evenwhen integrated into the chromosome of a host cell or present in anucleic acid solution with other DNA molecules.

As used herein, the term “protein-coding DNA molecule” refers to a DNAmolecule comprising a nucleotide sequence that encodes a protein. A“protein-coding sequence” means a DNA sequence that encodes a protein. A“sequence” means a sequential arrangement of nucleotides or amino acids.The boundaries of a protein-coding sequence may be determined by atranslation start codon at the 5′-terminus and a translation stop codonat the 3′-terminus. A protein-coding molecule may comprise a DNAsequence encoding a protein sequence. As used herein, “transgeneexpression”, “expressing a transgene”, “protein expression”, and“expressing a protein” mean the production of a protein through theprocess of transcribing a DNA molecule into messenger RNA (mRNA) andtranslating the mRNA into polypeptide chains, which are ultimatelyfolded into proteins. A protein-coding DNA molecule may be operablylinked to a heterologous promoter in a DNA construct for use inexpressing the protein in a cell transformed with the recombinant DNAmolecule. As used herein, “operably linked” means two DNA moleculeslinked in manner so that one may affect the function of the other.Operably-linked DNA molecules may be part of a single contiguousmolecule and may or may not be adjacent. For example, a promoter isoperably linked with a protein-coding DNA molecule in a DNA constructwhere the two DNA molecules are so arranged that the promoter may affectthe expression of the transgene.

As used herein, a “DNA construct” is a recombinant DNA moleculecomprising two or more heterologous DNA sequences. DNA constructs areuseful for transgene expression and may be comprised in vectors andplasmids. DNA constructs may be used in vectors for the purpose oftransformation, that is the introduction of heterologous DNA into a hostcell, to produce transgenic plants and cells, and as such may also becontained in the plastid DNA or genomic DNA of a transgenic plant, seed,cell, or plant part. As used herein, a “vector” means any recombinantDNA molecule that may be used for the purpose of bacterial or planttransformation. Recombinant DNA molecules as set forth in the sequencelisting, can, for example, be inserted into a vector as part of aconstruct having the recombinant DNA molecule operably linked to a geneexpression element that functions in a plant to affect expression of theengineered protein encoded by the recombinant DNA molecule. Generalmethods useful for manipulating DNA molecules for making and usingrecombinant DNA constructs and plant transformation vectors are wellknown in the art and described in detail in, for example, handbooks andlaboratory manuals including M R Green and J Sambrook, “MolecularCloning: A Laboratory Manual” (Fourth Edition) ISBN:978-1-936113-42-2,Cold Spring Harbor Laboratory Press, NY (2012). The components for a DNAconstruct, or a vector comprising a DNA construct, include one or moregene expression elements operably linked to a transcribable DNAsequence, such as the following: a promoter for the expression of anoperably linked DNA, an operably linked protein-coding DNA molecule, andan operably linked 3′ untranslated region (UTR). Gene expressionelements useful in practicing the present invention include, but are notlimited to, one or more of the following type of elements: promoter, 5′UTR, enhancer, leader, cis-acting element, intron, targeting sequence,3′ UTR, and one or more selectable marker transgenes.

The DNA constructs of the invention may include a promoter operablylinked to a protein-coding DNA molecule provided by the invention,whereby the promoter drives expression of the recombinant proteinmolecule. Promoters useful in practicing the present invention includethose that function in a cell for expression of an operably linkedpolynucleotide, such as a bacterial or plant promoter. Plant promotersare varied and well known in the art and include, for instance, thosethat are inducible, viral, synthetic, constitutive, temporallyregulated, spatially regulated, and/or spatio-temporally regulated.

In one embodiment of the invention, a DNA construct provided hereinincludes a targeting sequence that is operably linked to a heterologousnucleic acid encoding a polypeptide molecule that hasherbicide-insensitive protoporphyrinogen oxidase activity, whereby thetargeting sequence facilitates localizing the polypeptide moleculewithin the cell. Targeting sequences are known in the art as signalsequences, targeting peptides, localization sequences, and transitpeptides. An example of a targeting sequence is a chloroplast transitpeptide (CTP), a mitochondrial targeting sequence (MTS), or a dualchloroplast and mitochondrial targeting peptide. By facilitating proteinlocalization within the cell, the targeting sequence may increase theaccumulation of recombinant protein, protect the protein fromproteolytic degradation, and/or enhance the level of herbicidetolerance, and thereby reduce levels of injury in the transgenic cell,seed, or organism after herbicide application.

CTPs and other targeting molecules that may be used in connection withthe present invention are known in the art and include, but are notlimited to, the Arabidopsis thaliana EPSPS CTP (Klee et al., Mol GenGenet. 210:437-442, 1987), the Petunia hybrida EPSPS CTP (della-Cioppaet al., PNAS 83:6873-6877, 1986), the maize cab-m7 signal sequence(Becker et al., Plant Mol Biol. 20:49-60, 1992; PCT WO 97/41228), amitochondrial pre-sequence (e.g. Silva Filho et al., Plant Mol Biol30:769-780, 1996), and the pea glutathione reductase signal sequence(Creissen et al., Plant J. 8:167-175, 1995; PCT WO 97/41228).

Recombinant DNA molecules of the present invention may be synthesizedand modified by methods known in the art, either completely or in part,where it is desirable to provide sequences useful for DNA manipulation(such as restriction enzyme recognition sites or recombination-basedcloning sites), plant-preferred sequences (such as plant-codon usage orKozak consensus sequences), or sequences useful for DNA construct design(such as spacer or linker sequences). The present invention includesrecombinant DNA molecules and engineered proteins having at least 70%sequence identity, at least 80% sequence identity, at least 85% sequenceidentity, at least 90% sequence identity, at least 95% sequenceidentity, at least 96% sequence identity, at least 97% sequenceidentity, at least 98% sequence identity, and at least 99% sequenceidentity to any of the recombinant DNA molecule or polypeptide sequencesprovided herein, and having herbicide-insensitive protoporphyrinogenoxidase activity. As used herein, the term “percent sequence identity”or “% sequence identity” refers to the percentage of identicalnucleotides or amino acids in a linear polynucleotide or polypeptidesequence of a reference (“query”) sequence (or its complementary strand)as compared to a test (“subject”) sequence (or its complementary strand)when the two sequences are optimally aligned (with appropriatenucleotide or amino acid insertions, deletions, or gaps totaling lessthan 20 percent of the reference sequence over the window ofcomparison). Optimal alignment of sequences for aligning a comparisonwindow are well known to those skilled in the art and may be conductedby tools such as the local homology algorithm of Smith and Waterman, thehomology alignment algorithm of Needleman and Wunsch, the search forsimilarity method of Pearson and Lipman, and by computerizedimplementations of these algorithms such as GAP, BESTFIT, FASTA, andTFASTA available as part of the Sequence Analysis software package ofthe GCG® Wisconsin Package® (Accelrys Inc., San Diego, Calif.), MEGAlign(DNAStar Inc., 1228 S. Park St., Madison, Wis. 53715), and MUSCLE(version 3.6) (Edgar, “MUSCLE: multiple sequence alignment with highaccuracy and high throughput” Nucleic Acids Research 32(5):1792-7(2004)) for instance with default parameters. An “identity fraction” foraligned segments of a test sequence and a reference sequence is thenumber of identical components that are shared by the two alignedsequences divided by the total number of components in the portion ofthe reference sequence segment being aligned, that is, the entirereference sequence or a smaller defined part of the reference sequence.Percent sequence identity is represented as the identity fractionmultiplied by 100. The comparison of one or more sequences may be to afull-length sequence or a portion thereof, or to a longer sequence.

Engineered proteins may be produced by changing (that is, modifying) awild-type protein to produce a new protein with modifiedcharacteristic(s) e.g. a particular cellular localization pattern, suchas targeted to the chloroplast or mitochondria, or a novel combinationof useful protein characteristics, such as altered V_(max), K_(m),K_(i), IC₅₀, substrate specificity, inhibitor/herbicide specificity,substrate selectivity, the ability to interact with other components inthe cell such as partner proteins or membranes, and protein stability,among others. Modifications may be made at specific amino acid positionsin a protein and may be a substitution of the amino acid found at thatposition in nature (that is, in the wild-type protein) with a differentamino acid. Engineered proteins provided by the invention thus provide anew protein with one or more altered protein characteristics relative toa similar protein found in nature. In one embodiment of the invention,an engineered protein has altered protein characteristics, such as thosethat result in decreased sensitivity to one or more herbicides ascompared to a similar wild-type protein, or improved ability to conferherbicide tolerance on a transgenic plant expressing the engineeredprotein to one or more herbicides. In one embodiment, the inventionprovides an engineered protein, and the recombinant DNA moleculeencoding it, comprising at least one amino acid substitution selectedfrom Table 1 and having at least about 70% sequence identity, about 80%sequence identity, about 85% sequence identity, about 90% sequenceidentity, about 95% sequence identity, about 96% sequence identity,about 97% sequence identity, about 98% sequence identity, and about 99%sequence identity to any of the engineered protein sequences providedherein, including but not limited to SEQ ID NO:1-20. Amino acidmutations may be made as a single amino acid substitution in the proteinor in combination with one or more other mutation(s), such as one ormore other amino acid substitution(s), deletions, or additions.Mutations may be made by any method known to those of skill in the art.

TABLE 1 Amino Acid Substitutions. Conservative Conservative ResidueSubstitutions Residue Substitutions Ala Ser Leu Ile; Val Arg Lys LysArg; Gln Asn Gln; His Met Leu; Ile Asp Glu Phe Met; Leu; Tyr Gln Asn SerThr; Gly Cys Ser Thr Ser; Val Glu Asp Trp Tyr Gly Pro Tyr Trp; Phe HisAsn; Gln Val Ile; Leu Ile Leu; Val

As used herein, “wild-type” means a naturally occurring similar, but notidentical, version. A “wild-type DNA molecule” or “wild-type protein” isa naturally occurring version of the DNA molecule or protein, that is, aversion of the DNA molecule or protein pre-existing in nature. Anexample of a wild-type protein useful for comparison with the engineeredproteins provided by the invention is the protoporphyrinogen oxidasefrom Arabidopsis thaliana. A “wild-type plant” is a non-transgenic plantof the same type as the transgenic plant, and as such is geneticallydistinct from the transgenic plant comprising the herbicide tolerancetrait. Examples of a wild-type plant useful for comparison withtransgenic maize plants are non-transgenic LH244 maize (ATCC depositnumber PTA-1173) and 01DKD2 inbred maize (I294213) (ATCC deposit numberPTA-7859). For transgenic soybean plants an exemplary comparative linewould be non-transgenic A3555 soy (ATCC deposit number PTA-10207), andfor transgenic cotton plants an exemplary comparative line would benon-transgenic Coker 130 (Plant Variety Protection Number 8900252).

Transgenic Plants & Herbicides

One aspect of the invention includes transgenic plant cells, transgenicplant tissues, transgenic plants, and transgenic seeds that comprise therecombinant DNA molecules and engineered proteins provided by theinvention. These cells, tissues, plants, and seeds comprising therecombinant DNA molecules and engineered proteins exhibit herbicidetolerance to one or more PPO herbicide(s), and, optionally, tolerance toone or more additional herbicide(s).

Suitable methods for transformation of host plant cells for use with thecurrent invention include virtually any method by which DNA can beintroduced into a cell (for example, where a recombinant DNA constructis stably integrated into a plant chromosome) and are well known in theart. An exemplary and widely utilized method for introducing arecombinant DNA construct into plants is the Agrobacteriumtransformation system, which is well known to those of skill in the art.Another exemplary method for introducing a recombinant DNA constructinto plants is insertion of a recombinant DNA construct into a plantgenome at a pre-determined site by methods of site-directed integration.Site-directed integration may be accomplished by any method known in theart, for example, by use of zinc-finger nucleases, engineered or nativemeganucleases, TALE-endonucleases, or an RNA-guided endonuclease (forexample a CRISPR/Cas9 system). Transgenic plants can be regenerated froma transformed plant cell by the methods of plant cell culture. Atransgenic plant homozygous with respect to a transgene (that is, twoallelic copies of the transgene) can be obtained by self-pollinating(selfing) a transgenic plant that contains a single transgene allelewith itself, for example an R0 plant, to produce R1 seed. One fourth ofthe R1 seed produced will be homozygous with respect to the transgene.Plants grown from germinating R1 seed can be tested for zygosity, usinga SNP assay, DNA sequencing, or a thermal amplification assay thatallows for the distinction between heterozygotes and homozygotes,referred to as a zygosity assay.

As used herein, a “PPO inhibitor herbicide” or “PPO herbicide” is achemical that targets and inhibits the enzymatic activity of aprotoporphyrinogen oxidase (PPO), which catalyzes the dehydrogenation ofprotoporphyrinogen IX to form protoporphyrin IX, which is the precursorto heme and chlorophyll. Inhibition of protoporphyrinogen oxidase causesformation of reactive oxygen species, resulting in cell membranedisruption and ultimately the death of susceptible cells. PPO herbicidesare well-known in the art and commercially available. Examples of PPOherbicides include, but are not limited to, diphenylethers (such asacifluorfen, its salts and esters, aclonifen, bifenox, its salts andesters, ethoxyfen, its salts and esters, fluoronitrofen, furyloxyfen,halosafen, chlomethoxyfen, fluoroglycofen, its salts and esters,lactofen, its salts and esters, oxyfluorfen, and fomesafen, its saltsand esters); thiadiazoles (such as fluthiacet-methyl and thidiazimin);pyrimidinediones or phenyluracils (such as benzfendizone, butafenacil,ethyl [3-2-chloro-4-fluoro-5-(1-methyl-6-trifluoro methyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-3-yl)phenoxy]-2-pyridyloxy] acetate (CASRegistry Number 353292-31-6 and referred to herein as S-3100),flupropacil, saflufenacil, and tiafenacil); phenylpyrazoles (such asfluazolate, pyraflufen and pyraflufen-ethyl); oxadiazoles (such asoxadiargyl and oxadiazon); triazolinones (such as azafenidin,bencarbazone, carfentrazone, its salts and esters, and sulfentrazone);oxazolidinediones (such as pentoxazone); N-phenylphthalimides (such ascinidon-ethyl, flumiclorac, flumiclorac-pentyl, and flumioxazin);benzoxazinone derivatives (such as 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3,4-dihydro-3-oxo-4-prop-2-ynyl-2H-1,4-benzoxazin-6-yl)-1,3,5-triazinane-2,4-dione);flufenpyr and flufenpyr-ethyl; pyraclonil; and profluazol.Protoporphyrinogen oxidases and cells, seeds, plants, and plant partsprovided by the invention exhibit herbicide tolerance to one or more PPOherbicide(s).

Herbicides may be applied to a plant growth area comprising the plantsand seeds provided by the invention as a method for controlling weeds.Plants and seeds provided by the invention comprise an herbicidetolerance trait and as such are tolerant to the application of one ormore PPO herbicides. The herbicide application may be the recommendedcommercial rate (1×) or any fraction or multiple thereof, such as twicethe recommended commercial rate (2×). Herbicide rates may be expressedas acid equivalent per pound per acre (lb ae/acre) or acid equivalentper gram per hectare (g ae/ha) or as pounds active ingredient per acre(lb ai/acre) or grams active ingredient per hectare (g ai/ha), dependingon the herbicide and the formulation. The herbicide applicationcomprises at least one PPO herbicide. The plant growth area may or maynot comprise weed plants at the time of herbicide application. Aherbicidally effective dose of PPO herbicide(s) for use in an area forcontrolling weeds may consist of a range from about 0.1× to about 30×label rate(s) over a growing season. The 1× label rate for someexemplary PPO herbicides is provided in Table 2. One (1) acre isequivalent to 2.47105 hectares and one (1) pound is equivalent to453.592 grams. Herbicide rates can be converted between English andmetric as: (lb ai/ac) multiplied by 1.12=(kg ai/ha) and (kg ai/ha)multiplied by 0.89=(lb ai/ac).

TABLE 2 Exemplary PPO Herbicides PPO Herbicide Chemical Family 1X Rateacifluorfen Diphenylethers 420 g ai/ha fomesafen Diphenylethers 420 gai/ha lactofen Diphenylethers 7-220 g ai/ha fluoroglycofen-ethylDiphenylethers 15-40 g ai/ha oxyfluorfen Diphenylethers 0.28-2.24 kgai/ha flumioxazin N-phenylphthalimide 70 g ai/ha azafenidin Triazolinone240 g ai/ha carfentrazone-ethyl Triazolinone 4-36 g ai/ha sulfentrazoneTriazolinone 0.1-0.42 kg ai/ha fluthiacet-methyl Thiadiazole 3-15 gai/ha oxadiargyl Oxadiazole 50-150 g ai/ha oxadiazon Oxadiazole2.24-4.48 kg ai/ha pyraflufen-ethyl Phenylpyrazole 6-12 g ai/hasaflufenacil Pyrimidine dione 25-50 g/ha S-3100 Pyrimidine dione 5-80g/ha

Herbicide applications may be sequentially or tank mixed with one, two,or a combination of several PPO herbicides or any other compatibleherbicide. Multiple applications of one herbicide or of two or moreherbicides, in combination or alone, may be used over a growing seasonto areas comprising transgenic plants of the invention for the controlof a broad spectrum of dicot weeds, monocot weeds, or both, for example,two applications (such as a pre-planting application and apost-emergence application or a pre-emergence application and apost-emergence application) or three applications (such as apre-planting application, a pre-emergence application, and apost-emergence application or a pre-emergence application and twopost-emergence applications).

As used herein, “tolerance” or “herbicide tolerance” means a plant,seed, or cell's ability to resist the toxic effects of an herbicide whenapplied. Herbicide tolerant crops can continue to grow and areunaffected or minimally affected by the presence of the appliedchemical. As used herein, an “herbicide tolerance trait” is a transgenictrait imparting improved herbicide tolerance to a plant as compared tothe wild-type plant. Contemplated plants which might be produced with anherbicide tolerance trait of the present invention could include, forinstance, any plant including crop plants such as soybean (e.g. Glycinemax), corn (maize), cotton (Gossypium sp.), and canola, among others.

The transgenic plants, progeny, seeds, plant cells, and plant parts ofthe invention may also contain one or more additional traits. Additionaltraits may be introduced by crossing a plant containing a transgenecomprising the recombinant DNA molecules provided by the invention withanother plant containing one or more additional trait(s). As usedherein, “crossing” means breeding two individual plants to produce aprogeny plant. Two plants may thus be crossed to produce progeny thatcontain the desirable traits from each parent. As used herein “progeny”means the offspring of any generation of a parent plant, and transgenicprogeny comprise a DNA construct provided by the invention and inheritedfrom at least one parent plant. Additional trait(s) also may beintroduced by co-transforming a DNA construct for that additionaltransgenic trait(s) with a DNA construct comprising the recombinant DNAmolecules provided by the invention (for example, with all the DNAconstructs present as part of the same vector used for planttransformation) or by inserting the additional trait(s) into atransgenic plant comprising a DNA construct provided by the invention orvice versa (for example, by using any of the methods of planttransformation or genome editing on a transgenic plant or plant cell).Such additional traits include, but are not limited to, increased insectresistance, increased water use efficiency, increased yield performance,increased drought resistance, increased seed quality, improvednutritional quality, hybrid seed production, and herbicide tolerance, inwhich the trait is measured with respect to a wild-type plant. Exemplaryadditional herbicide tolerance traits may include transgenic ornon-transgenic tolerance to one or more herbicides such as ACCaseinhibitors (for example aryloxyphenoxy propionates andcyclohexanediones), ALS inhibitors (for example sulfonylureas,imidazolinones, triazoloyrimidines, and triazolinones) EPSPS inhibitors(for example glyphosate), synthetic auxins (for example phenoxys,benzoic acids, carboxylic acids, semicarbazones), photosynthesisinhibitors (for example triazines, triazinones, nitriles,benzothiadiazoles, and ureas), glutamine synthesis inhibitors (forexample glufosinate), HPPD inhibitors (for example isoxazoles,pyrazolones, and triketones), PPO inhibitors (for examplediphenylethers, N-phenylphthalimide, aryl triazinones, andpyrimidinediones), and long-chain fatty acid inhibitors (for examplechloroacetamindes, oxyacetamides, and pyrazoles), among others.Exemplary insect resistance traits may include resistance to one or moreinsect members within one or more of the orders of Lepidoptera,Coleoptera, Hemiptera, and Homoptera, among others. Such additionaltraits are well known to one of skill in the art; for example, and alist of such transgenic traits is provided by the United StatesDepartment of Agriculture's (USDA) Animal and Plant Health InspectionService (APHIS).

A cell transformed with a polynucleotide of the present invention, suchas an expression construct, may be selected for the presence of thepolynucleotide or its encoded enzymatic activity before or afterregenerating such a cell into a transgenic plant. Transgenic plantscomprising such a polynucleotide may thus be selected for instance byidentifying a transgenic plant that comprises the polynucleotide or theencoded enzymatic activity, and/or displays an altered trait relative toan otherwise isogenic control plant. Such a trait may be, for example,tolerance to a PPO herbicide.

Transgenic plants and progeny that contain a transgenic trait providedby the invention may be used with any breeding methods that are known inthe art. In plant lines comprising two or more transgenic traits, thetransgenic traits may be independently segregating, linked, or acombination of both in plant lines comprising three or more transgenictraits. Back-crossing to a parental plant and out-crossing with anon-transgenic plant are also contemplated, as is vegetativepropagation. Descriptions of breeding methods that are used fordifferent traits and crops are well known to those of skill in the art.To confirm the presence of the transgene(s) in a particular plant orseed, a variety of assays may be performed. Such assays include, forexample, molecular biology assays, such as Southern and northernblotting, PCR, and DNA sequencing; biochemical assays, such as detectingthe presence of a protein product, for example, by immunological means(ELISAs and western blots) or by enzymatic function; plant part assays,such as leaf or root assays; and also, by analyzing the phenotype of thewhole plant.

Introgression of a transgenic trait into a plant genotype is achieved asthe result of the process of backcross conversion. A plant genotype intowhich a transgenic trait has been introgressed may be referred to as abackcross converted genotype, line, inbred, or hybrid. Similarly a plantgenotype lacking the desired transgenic trait may be referred to as anunconverted genotype, line, inbred, or hybrid.

Having described the invention in detail, it will be apparent thatmodifications, variations, and equivalent embodiments are possiblewithout departing the scope of the invention defined in the appendedclaims. Furthermore, it should be appreciated that the examples in thepresent disclosure are provided as non-limiting examples.

EXAMPLES

The following examples are included to demonstrate embodiments of theinvention. It should be appreciated by those of skill in the art thatthe techniques disclosed in the examples that follow representtechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the concept, spirit, andscope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein with the same or similarresult achieved. All such similar substitutes and modifications apparentto those skilled in the art are deemed to be within the spirit, scope,and concept of the invention as defined by the appended claims.

Example 1 Microbial Protoporphyrinogen Oxidase Discovery

Novel protoporphyrinogen oxidases were identified from microbialsequence databases using bioinformatic methods and a novelprotoporphyrinogen oxidase bacterial screening system. The sequence ofE. coli HemG (SEQ ID NO:4) was use as a starting sequence forbioinformatic analysis of microbial sequence databases. Thebioinformatic analysis identified thirty-three novel putativeprotoporphyrinogen oxidases of the HemG PPO family from diversebacterial sources. The sequences encoding these putative HemG PPOenzymes were compared using phylogenetic tree mapping and found to berelatively diverse. Ten were selected for further analysis from thisgroup of thirty-three due to their representation of individual uniqueclustered members on the phylogenetic tree.

The coding sequences for the ten selected HemG PPO enzymes wereoptimized for E. coli expression to eliminate any rare codons found inthe wild-type DNA sequence. The E. coli optimized coding sequences forthe ten HemG PPO enzymes were then cloned into bacterial expressionvectors. An herbicide-sensitive PPO enzyme found naturally in waterhemp(Amaranthus tuberculatus) was cloned into a bacterial expression vectorfor use as a control for both PPO function and herbicide-sensitivity(referred to as “WH” and provided as SEQ ID NO:21; NCBI GenBankAccession No. ABD52326; Patzoldt, et al. “A codon deletion confersresistance to herbicides inhibiting protoporphyrinogen oxidase”Proceedings of the National Academy of Science USA. 103(33):12329-12334(2006)). The waterhemp PPO enzyme is not a HemG family member. The HemGPPO enzyme found naturally in E. coli was cloned into a bacterialexpression vector for use as a control for both PPO activity and toassay it for herbicide-sensitivity (referred to as H_N10 and provided asSEQ ID NO:4).

A protoporphyrinogen oxidase bacterial screening system was created totest recombinant proteins for protoporphyrinogen oxidase activity andthus confirm that they are functional PPO enzymes. This screening systemused a functional rescue assay in an E. coli strain that contained agene knockout for the E. coli PPO enzyme (SEQ ID NO:4). The hemGknockout E. coli strain which was utilized showed very minimal growth onheme-free bacterial medium (such as LB medium), but growth was recoveredwhen the bacterial medium was supplemented with free heme or when anactive recombinant protoporphyrinogen oxidase was expressed in the E.coli. The hemG knockout E. coli strain could thus be used withrecombinant protein expression to quickly and easily assay proteins forprotoporphyrinogen oxidase activity.

The hemG knockout E. coli strain was transformed with bacterialexpression vectors containing the gene encoding the ten putative HemGPPO enzymes, the E. coli HemG PPO enzyme, and the waterhemp PPO enzyme.The bacteria were then streaked onto LB medium plates, which is aheme-free bacterial medium. Expression of a recombinant PPO enzymerescued the growth of E. coli, resulting in growth on the LB plates.Growth of the transformed hemG knockout E. coli strain on LB platesindicated that a protein sequence functioned as a protoporphyrinogenoxidase. Ten HemG PPO enzymes (SEQ ID NOs:1-10), and the waterhemp PPOenzyme (SEQ ID NO:21) were able to restore growth of the transformedhemG knockout E. coli strain in this assay, thus confirming their PPOactivity. An alignment of the protein sequences of PPO enzymes, withconsensus positions, is provided as FIG. 1. Using this assay, a largenumber of novel or engineered proteins can be screened to confirm andmeasure protoporphyrinogen oxidase activity.

Example 2 Protoporphyrinogen Oxidase Inhibitor Insensitivity

Novel protoporphyrinogen oxidases that are tolerant to PPO herbicideswere identified using an herbicide bacterial screening system. Thisscreening system used a growth assay of the hemG knockout E. coli strainin LB liquid medium with a PPO herbicide to identify protoporphyrinogenoxidases that were not sensitive to the PPO herbicide.

The hemG knockout E. coli strain was transformed with a bacterialexpression vector containing the confirmed protoporphyrinogen oxidaseactivity and cultured in LB liquid medium. Purified crystalline form ofone of five different PPO herbicides (acifluorfen (1 mM), flumioxazin(0.5 mM), lactofen (0.5 mM), fomesafen (1 mM), and S-3100 (100 uM),representing three different PPO chemistry subclasses, was added to themedium. Recombinant proteins were expressed and the E. coli growth rateswere measured. Growth curves (OD600) were measured for the differentvariants in the presence and absence of the PPO herbicides at selectedtime-points from time zero to twenty-four hours. The growth of atransformed hemG knockout E. coli strain in LB medium in the presence ofa PPO herbicide indicated that the gene used to transform the E. coliencoded an herbicide-insensitive protoporphyrinogen oxidase (iPPO).

Novel protoporphyrinogen oxidases were used with this assay to test forinsensitivity to PPO herbicides. The ten protoporphyrinogen oxidasesprovided as SEQ ID NO: 1 through SEQ ID NO:10 were all found to confernormal growth rates on the hemG knockout E. coli strain in LB medium inthe presence of a PPO herbicide, indicating that these proteins areherbicide-insensitive protoporphyrinogen oxidases (iPPO) (FIG. 2). ThehemG knockout E. coli strain expressing the waterhemp PPO (SEQ ID NO:21)was sensitive to all five PPO herbicides, confirming that the assay wasable to distinguish between sensitive and insensitive protoporphyrinogenoxidases for each of the herbicides. Using this assay, a large number ofnovel or engineered proteins can be screened to confirmprotoporphyrinogen oxidase activity in the presence of PPO herbicide(s).

Example 3 Protoporphyrinogen Oxidase (PPO) Enzyme Assay

Protoporphyrinogen oxidases were enzymatically characterized to measureeach PPO enzyme's substrate binding affinity (K_(m)) and sensitivity tothe PPO herbicide (IC50). Wild-type plant PPO enzymes from waterhemp,soybean, and maize were used for comparison to the microbial HemGprotoporphyrinogen oxidases (provided as SEQ ID NO: 1 through SEQ IDNO:10).

Etioplasts and chloroplasts were prepared from etiolated cotyledons(soybean, Glycine max), etiolated leaves/coleoptiles (maize, Zea mays)and unfolded apical leaves (waterhemp, Amaranthus tuberculata) generallyby the procedure described by Grossmann et al., (“The HerbicideSaflufenacil (Kixor™) is a New Inhibitor of Protoporphyrinogen IXOxidase Activity” Weed Science, 58(1):1-9 (2010)). Soybean (A3555) andmaize (LH244) seeds were placed between two sheets of moist germinationpaper (Anchor Paper Company, Saint Paul, Minn., USA) in a beaker ofwater in continuous darkness for eight to ten days. Waterhemp plantswere grown for 30 days in the greenhouse. Tissue was collected, placedbetween moist sheets of paper towels until ground to fine powder with amortar and pestle in liquid nitrogen. Homogenization buffer (50 mMTris-HCl, pH 7.4, 500 mM sucrose, 1 mM EDTA, 1 mM magnesium chloride,and 2 g/liter bovine serum albumin) was added to the frozen powder at4:1 (ml homogenization buffer to g fresh weight tissue), mixedvigorously and filtered through four layers of pre-moistened Miracloth™(Merck-Millipore, Darmstadt, Germany). The filtrate was centrifuged at9299 g for five minutes. The pellet was re-suspended in homogenizationbuffer and centrifuged at 150 g for two minutes. The supernatantsolution was centrifuged at 4000 g for fifteen minutes. Allcentrifugation steps were carried out a 4° C. The pellet (intact plastidfraction) was re-suspended in 50 mM Tris-HCl (pH 7.4), 2 mM EDTA and 20%(v/v) glycerin and stored in aliquots at −80° C. Total protein inplastid preparations was measured by the Bradford method (M M Bradford,“A rapid and sensitive method for the quantitation of microgramquantities of protein utility the principle of protein-dye binding”Analytical Biochemistry, 72:248-254 (1976)) with bovine serum albumin asthe standard.

Selected PPO enzymes were expressed in the E. coli hemG knockout cellline. Bacterial cells from an overnight culture were used to inoculate20 ml of fresh media. These cultures were allowed to grow forapproximately 48 hrs at 20° C. to a dense culture. Bacterial cells werecollected by centrifugation and the cell pellets stored at −80° C. untilenzyme assays were performed. Frozen bacterial pellets were re-suspendedin extraction buffer (50 mM Tris-HCl, pH 7.6, 1 mM EDTA & 1 mM MgCl₂)and sonicated (Sonics VibraCell™, Newtown, Conn. USA) for three cyclesof 30 seconds in an ice bath with a one-minute rest period betweencycles. Broken cells were centrifuged at 200 g for 2 minutes at 4° C.and the supernatant solution was used for PPO enzyme assays afterdilution with extraction buffer. Total protein was measured by themethod of Bradford (1976) with bovine serum albumin as the standard.

Protoporphryrinogen IX (protogen) was synthesized by reduction ofcommercially-available protoporphyrin with sodium mercury amalgam asdescribed by J M Jacobs and N J Jacobs (“Measurement ofProtoporphyrinogen Oxidase Activity” in Current Protocols in Toxicology(1999) 8.5.1-8.5.13, John Wiley & Sons, Inc.). Protoporphyrin (Proto)was added to 0.01N potassium hydroxide in 20% ethanol and stirred in thedark until dissolved (about 40 minutes). A volume of 0.8 ml of wasplaced in a 2-ml polypropylene vial with a screw-top cap containing anO-ring, and about 1 g (a spatula tipful, oil drained off) of sodiummercury amalgam (Product Number 451908, Sigma-Aldrich, St. Louis, Mo.,stored under oil) was added. The tube was immediately capped and mixedvigorously with a vortex mixer and vented about every 30 seconds byloosening the cap until the solution was no longer fluorescing red undera UV light (about five minutes). The reaction vial was flushed withargon and centrifuged briefly to pellet the remaining sodium amalgam.The supernatant solution was immediately diluted 1:1 (v/v) with asolution of 0.1M DTT and 0.5M Tris-HCl, pH 7.5 and the vial flushed withargon. The resulting solution was split into smaller aliquots into 0.5ml polypropylene capped tubes which were flushed with argon immediatelyafter the aliquot was added. Capped tubes were covered with aluminumfoil and stored at −80° C. For the enzyme assay, the protogen aliquotswere thawed, stored covered on ice, and used on the same day. Theconcentration of protogen in the preparation was calculated bysubtracting the Proto concentration, as measured by fluorescence HPLC(method described by Matsumoto et al, “Porphyrin Intermediate Involvedin Herbicidal Action of delta-Aminolevulinic Acid on Duckweed (Lemnapaucicostata Hegelm.)” Pesticide Biochem. and Phys. 48:214-221 (1994)),in the final protogen solution (typically about 1% of starting material)from the Proto concentration in the starting material and assuming nosignificant impurities in either sample. Protogen prepared and storedunder these conditions was stable for at least six months.

PPO activity in plant plastid extract and bacterial extract preparationswas measured generally as described by Grossmann. et al. (2010). Tenmicroliters of either plastid extract (40 μg total protein) or bacterialextract (16 to 35 μg total protein for the different bacterial extracts)was added to assay buffer (100 mM Tris-HCl, pH 7.4, 5 mM DTT, 1 mM EDTAand 0.085% (v/v) Tween 80). S-3100 (also known as “SYN-523”; e.g. U.S.Patent Publication US20100062941 A1) was added as a two-microlitervolume from a 100× stock solution prepared in acetone. Analytical-gradeS-3100 was provided by Sumitomo Chemical Company. All assays were run ina final concentration of 1% (v/v) acetone. The extracts (plastid orbacterial), buffer, and S-3100 were incubated at 30° C. (plant extracts)or 37° C. (bacterial extracts) for five minutes before addition of twomicroliters of protogen to initiate the assay. All assays were done in a96-well black polystyrene microtiter plate (Costar® 3925, Corning, Inc.,Corning, N.Y.) at a final volume of 200 microliters. After protogenaddition (3 μM for IC₅₀ measurements; variable for K_(m) measurements)to all wells, the plate was incubated at 30° C. (plant extracts) or 37°C. (bacterial extracts) before initiating data collection. Fluorescenceover time was measured at 30° C. (plant extracts) or 37° C. (bacterialextracts) with excitation and emission wavelengths of 405 mm and 630 mm,respectively, in a SpectraMax® M5 Multi-Mode Microplate Reader(Molecular Devices, Sunnyvale, Calif.). An assay blank was run by addingheat-inactivated (five minutes at 100° C.) extract to the assay mixture.

Substrate (protoporphyrinogen) binding affinity of protoporphyrinogenoxidases was measured as the K_(m). The apparent K_(m) values for eachPPO evaluated were calculated using rectangular hyperbola curve-fittingusing the SoftPro® kinetics software package (Molecular Devices,Sunnyvale, Calif.). Enzyme activity sensitivity to the PPO herbicideS-3100 was measured as the concentration giving 50% inhibition ofcontrol activity (IC₅₀). The S-3100 IC₅₀ values for each PPO evaluatedwere determined graphically from the semi logarithmic plot of S-3100concentration versus PPO activity.

The K_(m) for the PPO enzymes from three plant sources (waterhemp,soybean, or maize) and the microbial HemG PPO enzymes (SEQ ID NO: 1through SEQ ID NO:10) were found to be similar, ranging from 0.3 uM to2.0 uM. The three plant PPO enzymes tested were sensitive to S-3100 withIC₅₀ values of 0.009, 0.004, and 0.003 uM, respectively. In contrast,the PPO enzymes from a bacterial source (SEQ ID NO: 1 through SEQ IDNO:10) were insensitive to S-3100 with IC₅₀ values greater than 100 uM.Data are provided in Table 3.

TABLE 3 PPO enzymatic activity of enzymes purified from plant ormicrobial sources Source K_(m) (uM) S-3100 IC₅₀ (uM) Waterhemp 0.7 0.009Soybean 1.8 0.004 Maize 2.0 0.003 H_N10 (SEQ ID NO: 4) 0.7 >100 H_N20(SEQ ID NO: 2) 0.8 >100 H_N30 (SEQ ID NO: 5) 1.0 >100 H_N40 (SEQ ID NO:6) 1.4 >100 H_N50 (SEQ ID NO: 7) 1.0 >100 H_N60 (SEQ ID NO: 3) 1.1 >100H_N70 (SEQ ID NO: 8) 1.4 >100 H_N90 (SEQ ID NO: 1) 1.2 >100 H_N100 (SEQID NO: 9) 0.6 >100 H_N110 (SEQ ID NO: 10) 0.3 >100

Example 4 Enzymatic Optimization of Protoporphyrinogen Oxidases

Protein optimization is used to improve or alter the enzymaticproperties of novel protoporphyrinogen oxidases. One or more methods ofprotein engineering are used to optimize the enzymes. Non-limitingexamples of protein engineering approaches include Alanine-ScanningMutations; Homology-Scanning Mutations; Pro/Gly Scanning Mutations;Region Swaps or Mutations; and combinations of these various techniques(see, M Lehmann and M Wyss, Current Opinion in Biotechnology12(4):371-375 (2001); B Van den Burg and VGH Eijsink, Current Opinion inBiotechnology 13(4):333-337 (2002); and Weiss et al., Proceedings of theNational Academy of Sciences USA 97(16):8950-8954 (2000)). DNA sequencesencoding engineered protoporphyrinogen oxidases are synthesized andcloned into the bacterial expression vector. The vector is used totransformed the hemG knockout E. coli strain for the initialhigh-throughput bacterial rescue screen as described in Example 1. Theengineered protoporphyrinogen oxidases that rescue the hemG knockout E.coli strain are screened for sensitivity to one or more PPO herbicide(s)using the bacterial growth assay as described in Example 2.Alternatively, the transformed hemG knockout E. coli strain is plated onmedium with and without a PPO herbicide. The engineered variants thatexhibit tolerance to PPO herbicides are expressed in a bacterialexpression system and a detailed biochemical characterization is doneusing the purified protein with the continuous fluorimetric assay asdescribed in Example 3. The engineered variants that are insensitive toPPO herbicides are selected for cloning into plant transformationvectors, plant transformation, and in planta testing.

Example 5 Expression and Testing of HemG PPO Enzymes in Maize

The microbial HemG PPO enzymes were expressed in transgenic maizeplants, and the transgenic plants were analyzed for PPO herbicidetolerance. Plant transformation vectors were constructed comprising arecombinant DNA molecule encoding one of the microbial HemG PPO enzymesprovided as SEQ ID NO: 1-20. The DNA sequence encoding a PPO enzyme caninclude at the 5′ end a codon for a methionine, commonly known as astart codon, or this codon can be eliminated to facilitate operablelinkage of a transit peptide sequence to the 5′ end of the codingsequence. Examples of PPO enzyme protein sequences containing amethionine at the amino-terminus are provided as SEQ ID NO:1-10.Examples of PPO enzyme protein sequences without a methionine at theamino-terminus are provided as SEQ ID NO:11-20. For planttransformation, the nucleotide sequences encoding the putative PPOenzymes were codon optimized for either dicot or monocot expression.Table 4 provides the SEQ ID NO corresponding to the protein andnucleotide sequences of the microbial HemG PPO enzymes in thetransformation vectors.

TABLE 4 SEQ ID NO corresponding to PPO variants Monocot Bacterial Dicotcodon codon PPO Protein DNA optimized optimized H_N10 4, 13 25 35, 44 57H_N20 2, 12 23 33, 43, 52 55 H_N30 5, 14 26 36, 45 58 H_N40 6, 15 27 37,46 59 H_N50 7, 16 28 38, 47 60 H_N60  3 24 34 56 H_N70  8 29 39 61 H_N901, 11, 18, 19 22 32, 42, 49, 50, 51 54 H_N100 9, 17, 20 30 40, 48, 53 62H_N110 10 31 41 63 Waterhemp 21 n/a n/a n/a PPO

Four plant transformation vectors were created for expressing the HemGPPO H_N10 (SEQ ID NO:4) using two different combinations of promoterplus leader plus intron with two different targeting peptide (TP)sequences and two different 3′UTR sequences. The plant transformationconstructs were annotated construct 1, 6, 11, and 16. For maize inplanta testing, immature maize (LH244) embryos were transformed withusing Agrobacterium tumefaciens and standard methods known in the art.

Transgenic maize plants were produced using the plant transformationconstructs 6 and 16 containing the gene encoding the HemG PPO H_N10 (SEQID NO:4). Leaf samples were collected from R0 plants and screened by PCRto determine the number of copies of the transgene inserted into theplant genome. The plants containing a single copy (single copy) wereselfed and outcrossed to generate R1 and F1 seed for future testing,respectively. Plants containing multiple copies (multi-copy) weresprayed as follows: (1) 5 g/ha S-3100 at approximately the V5 growthstage followed by 10 g/ha S-3100 at approximately the V7 growth stage,for a total application of 15 g/ha S-3100 or (2) 10 g/ha S-3100 atapproximately the V5 growth stage. The average percentage of injury, ona scale of 0-100 with zero being no injury and 100 being complete cropdeath, was assessed 7 days after the final treatment for each batch.Sprayed non-transgenic LH244 maize was used as a control and showed anaverage of 43.3% injury when treated with a total of 15 g/ha S-3100(Treatment 1) and an average injury of 26.7% when treated with a totalof 10 g/ha S-3100 (Treatment 2). Transgenic maize plants expressing HemGH_N10 (SEQ ID NO:4) outperformed the control plants with an average of28.9% injury when treated with a total of 15 g/ha S-3100 (Treatment 1)and an average injury of 24.2% when treated with a total of 10 g/haS-3100 (Treatment 2). Data are provided in Table 5.

TABLE 5 Transgenic Maize Herbicide Tolerance I Treatment ProteinConstruct Plants Tested Average Injury 1 n/a nontransgenic 12 43.3 1H_N10  6 18 28.9 2 n/a nontransgenic 6 26.7 2 H_N10 16 12 24.2

Transgenic maize plants were produced using the plant transformationconstructs 6, 20, 21, 22, and 23 containing the gene encoding the HemGPPO enzymes H_N90 (SEQ ID NO:1), H_N10 (SEQ ID NO:4), H_N60 (SEQ IDNO:3), H_N110 (SEQ ID NO:10), and H_N40 (SQ ID NO:6), respectively.These five constructs had the same promoter plus leader plus introncombination, with two different targeting peptide (TP) sequences, andthe same 3′UTR sequence. Leaf samples were taken from the R0 plants andanalyzed by PCR to determine the transgene copy number. Single copyplants for up to 12 unique events per construct were transplanted topots, selfed and outcrossed to generate R1 and F1 seed, respectively,for future testing. Multi-copy plants and extra single copy plants weresprayed with 40 grams/ha S-3100 at approximately the V5 growth stage andinjury ratings were taken 7 days after treatment. The average percent(%) injury for all plants expressing each PPO enzyme and the number ofplants that were deemed highly tolerant (less than 10% injury) wasnoted, where each plant is a unique single or multi-copy transformant.Transgenic maize plants expressing H_N10 (SEQ ID NO:4) had an overallaverage injury of 39.5% and produced 31 highly tolerant plants out of139 plants tested. Transgenic maize plants expressing H_N60 (SEQ IDNO:3) had an overall average injury of 55.8% and produced 19 highlytolerant plants out of the 86 plants tested. Transgenic maize plantsexpressing H_N90 (SEQ ID NO:1) had the lowest overall average injury at31.4% and produced 44 highly tolerant plants out of the 99 plantstested. Transgenic maize plants expressing H_N40 (SEQ ID NO:6) had anoverall average injury at 47.0% and produced the highest proportion ofhighly tolerant plants with 53 highly tolerant plants out of the 98plants tested. Transgenic maize plants expressing H_N110 (SEQ ID NO:10)had an average injury of 43.0% but did not produce any highly tolerantplants. Data are provided in Table 6.

TABLE 6 Transgenic Maize Herbicide Tolerance II SEQ ID Plants AverageHighly Construct Protein NO Tested Injury Tolerant 6 H_N10 4 139 39.5%31 20 H_N60 3 86 55.8% 19 21 H_N90 1 99 31.4% 44 22 H_N110 10 94 43.0% 023 H_N40 6 98 47.0% 53

The R0 transgenic maize data demonstrated that the five microbial HemGPPO enzymes H_N90 (SEQ ID NO:1), H_N10 (SEQ ID NO:4), H_N60 (SEQ IDNO:3), H_N40 (SEQ ID NO:6), and H₁₃ N110 (SQ ID NO:10) produced reducedinjury rates when expressed in transgenic plants and thus conferred croptolerance to a PPO herbicide.

Transgenic F1 plants produced from outcrossing the single copy R0 plantsexpressing H_N10 (SEQ ID NO:4) in one of two construct configurationswere tested in the greenhouse for herbicide tolerance. The plants weretreated with 40 grams/ha S-3100 at the V3 growth stage and injuryratings were taken seven days after treatment. Transgenic maize plantsexpressing H_N10 (SEQ ID NO:4) in the construct 6 configuration resultedin 13 out of 18 events producing highly tolerant plants (10% or lessinjury) but the construct 16 configuration resulted in no eventsproducing highly tolerant plants.

Transgenic F1 plants produced from outcrossing the single copy R0 plantsexpressing H_N10 (SEQ ID NO:4) in one of two construct configurations(constructs 6 and 16) were tested in the field for herbicide tolerance.This F1 population was segregating (50% hemizygous and 50% null) andselection for transgenic plants was not conducted prior to injuryratings. The overall average injury ratings for such a population areexpected to be higher than a homogenous transgenic population since itis difficult to discern non-transgenic plants from transgenic plants.The trials were conducted at two locations with two replicates and 3treatments per construct. Non-transgenic maize plants were used as anegative control. The herbicide application treatments were as follows:Treatment 1 was 0.036 lb ai/acre S-3100 applied at V2 followed by (fb)V4 fb V8; Treatment 2: was 0.072 lb ai/acre S-3100 applied at V2 fb V4fb V8; Treatment 3: was 0.144 lb ai/acre S-3100 applied at V2 fb V4 fbV8. Crop Injury Percent ratings were assessed at the V2 growth stage(CIPV2) and at the V4 growth stage (CIPV4) at 5 to 7 days aftertreatment (the error V2 and error V4 are half of the least significantdifference (LSD)). The crop injury ratings were combined for bothlocations. All non-transgenic plants and plants with events generatedusing construct 16 showed between 94.6-99.5% injury following both theV2 and V4 herbicide application for each of the three treatments. Plantswith events generated using construct 6 showed only 30% to 50% injuryfollowing the V2 herbicide application and no injury following the V4herbicide application. Data are provided in Table 7.

TABLE 7 Efficacy field trial of F1 maize containing H_N10 (SEQ ID NO: 4)Treatment Construct CIPV2 CIPV4 Error V2 Error V4 Trt 1 wildtype 94.6 998.6 1.2 6 37.5 0 8.6 1.2 16 96.3 98.5 8.6 1.2 Trt 2 wildtype 99.5 99.55.4 0 6 37.5 0 5.4 0 16 99.5 99.5 5.4 0 Trt 3 wildtype 99.5 99.5 0 0 650 0 0 0 16 99.5 99.5 0 0

The F1 transgenic maize greenhouse and field data demonstrated that themicrobial HemG PPO enzyme H_N10 (SEQ ID NO:4) produced reduced injuryrates when expressed in transgenic plants and thus conferred croptolerance to a PPO herbicide

Example 6 Expression and Testing of HemG PPO Enzymes in Soybean Plants

The microbial HemG PPO enzymes were expressed in transgenic soybeanplants, and the transgenic plants were analyzed for PPO herbicidetolerance. Plant transformation vectors were constructed comprising arecombinant DNA molecule encoding one of the microbial HemG PPO enzymesprovided as H_N10 (SEQ ID NO:4 and SEQ ID NO:13); H_N20 (SEQ ID NO:12);H_N30 (SEQ ID NO:14); H_N40 (SEQ ID NO:15); H_N50 (SEQ ID NO:16); H_N90(SEQ ID NO:11, 18, 19); and H_N100 (SEQ ID NO:17, 20).

In soybean, excised embryos (A3555) were transformed with thetransformation constructs 1 and 11 using A. tumefaciens and standardmethods known in the art. Transformation constructs 1 and 11 had thesame promoter plus leader plus intron combination, the same 3′UTRsequence, the same microbial HemG PPO H_(—l N)10 (SEQ ID NO:4), butdiffered in the targeting peptide (TP) sequences. Regenerated R0transgenic plantlets were grown in the greenhouse. Plants representingtwenty single copy R0 soybean events produced from construct 11 andexpressing the microbial PPO enzyme H_N10 (SEQ ID NO: 4) were sprayedwith 210 g/ha flumioxazin (Valor®, Valent U.S.A. Corporation, WalnutCreek Calif.). The herbicide was sprayed at the V3 developmental stagewith three fully developed trifoliate leaves and injury ratings wereassessed 8 days after treatment. The percentage of plants that weredeemed highly tolerant (15% or less injury) was noted. After applicationof 210 g/ha flumioxazin, the non-transgenic soybean control plants hadan average injury rating of 30% and no highly tolerant plants. Soybeanplants expressing the microbial PPO enzyme H_N10 (SEQ ID NO:4) had anaverage injury rating of 22%, and 9% of the plants tested were highlytolerant to the herbicide.

Plants representing ten multi-copy R0 soybean events produced fromconstruct 11 and expressing the microbial PPO enzyme H_N10 (SEQ ID NO:4) were sprayed with 5 g/ha S-3100. The herbicide was sprayed at the V3developmental stage with three fully developed trifoliate leaves andinjury ratings taken 8 days after treatment. The percentage of plantsthat were deemed highly tolerant (25% or less injury) was noted. Afterapplication of S-3100 (5 g/ha), the non-transgenic soybean controlplants had an average injury rating of 60% and no highly tolerantplants. Soybean plants expressing the microbial PPO enzyme H_N10 (SEQ IDNO: 4) had an average injury rating of 47%, and 30% of the plants testedwere highly tolerant to the herbicide.

Single-copy transgenic R1 soybean plants in the greenhouse were sprayedwith one of three herbicide application rates: Treatment 1 was 5 gramsai/ha S-3100 applied at V4 followed by (fb) R1; Treatment 2 was 10 gramsai/ha S-3100 applied at V4 fb R1; Treatment 3 was 30 grams ai/ha S-3100applied at V4 fb R1 . Crop Injury Percent at V2 (CIPV2) ratings wereassessed ten days after treatment. Non-transgenic plants had averageinjury ratings of 89% to 100% and were not available for rating at theR1 growth stage. The plants produced from construct 1 and expressing themicrobial PPO enzyme H_N10 (SEQ ID NO: 4) had injury ratings rangingfrom 3% to 15.7%. Data are provided in Table 8.

TABLE 8 Green house S-3100 efficacy screen of R1 soybean S-3100 % InjuryConstruct Treatment Rate V4 stage % Injury R1 stage 1 1  5 g/ha 4.2 3 12 10 g/ha 7.8 6.5 1 1 30 g/ha 9.4 15.7 Nontransgenic 1  5 g/ha 89Nontransgenic 2 10 g/ha 98 Nontransgenic 3 30 g/ha 100

A high-throughput plant transformation and screening method was used toevaluate large numbers of constructs in transgenic plants for herbicidetolerance in early transformation plantlet tissue. This allowed forfaster and higher volume screening of construct configurations and PPOenzymes.

The genes encoding the seven microbial HemG PPO enzymes H_N10 (SEQ IDNO:13); H_N20 (SEQ ID NO:12); H_N30 (SEQ ID NO:14); H_N40 (SEQ IDNO:15); H_N50 (SEQ ID NO:16); H_N90 (SEQ ID NO:11, 18, 19); and H_N100(SEQ ID NO:17, 20) were operably linked to thirty-seven differenttargeting peptides and cloned into a base plant transformation vector.This permitted the side-by-side comparison of seven different HemG PPOenzymes with thirty-seven different targeting peptides using the samepromoter and 3′UTR elements for gene expression. These planttransformation constructs were used to transform soybean excised embryos(germplasm A3555) using A. tumefaciens and standard methods known in theart. Four hundred explants were inoculated for each construct resultingin twelve containers per construct. A sterile PPO herbicide solution wasused for herbicide tolerance testing. The herbicide solution consistedof 0.3 g of S-3100 in crop oil concentrate (5.0 mL) and 495 mL ofdeionized water. This was filtered through a 0.45 micron Nalgene®Rapid-Flow™ Tissue Culture Filter Unit and Surfactant-Free CelluloseAcetate membrane filter unit (VWR, Radnor, Pa., USA). The resultingsterile solution was shaken before application.

At five weeks post-transformation, four of the twelve plant containersper construct were sprayed with two passes of the sterile PPO herbicidesolution. The treated plantlets were then enclosed in the container andreceived at least 15 hours of light exposure post spray each day forfour days. At the end of day four post application of S-3100, thetreated plantlets were photographed and scored on a visual scale ofgreen coloration (green coloration was representative of healthyphotosynthetic plant tissue as compared to photo-bleached tissue) versusdamage. The scoring values were 0 for poor tolerance, high damage, lowgreen coloration; 1 for some tolerance, average damage, moderate greencoloration; and 2 for good tolerance, low damage, high green coloration.The scoring for each construct is presented in Table 9, where n.d.indicates the analysis was not conducted. The results indicate that inthis high-throughput screening a number constructs provided tolerance tothe PPO herbicide.

TABLE 9 High-throughput soybean screening for herbicide tolerance:coloration score Targeting Peptide H_10 H_N20 H_N30 H_N40 H_N50 H_N90H_N100 TP1 n.d. 0 2 2 1 2 2 TP2 n.d. 0 0 1 1 2 1 TP3 1 1 n.d. 1 1 2 1TP4 n.d. 1 n.d. 2 1 1 n.d. TP5 n.d. n.d. n.d. n.d. 0 1 n.d. TP6 0 1 0n.d. 2 2 2 TP7 n.d. 2 1 2 1 2 2 TP8 0 1 1 2 1 0 0 TP9 1 1 1 0 1 1 0 TP10n.d. n.d. 0 n.d. 1 2 1 TP11 n.d. 1 1 1 0 2 0 TP12 1 2 1 1 1 2 2 TP13 1 21 1 1 2 0 TP14 0 0 n.d. 2 1 2 1 TP15 n.d. 1 1 2 2 2 2 TP16 1 1 n.d. n.d.0 1 1 TP17 n.d. 2 1 1 1 2 1 TP18 1 1 0 1 1 2 1 TP19 0 1 0 1 0 1 1 TP20 02 n.d. n.d. n.d. 2 2 TP21 0 0 n.d. 2 1 1 0 TP22 1 1 n.d. n.d. 2 2 2 TP231 1 n.d. 1 1 1 2 TP24 0 1 1 0 1 1 0 TP25 n.d. 1 n.d. 2 2 1 1 TP26 0 1 01 1 0 n.d. TP27 1 1 0 1 1 1 1 TP28 n.d. 1 0 1 1 2 1 TP29 n.d. 1 1 1 1 11 TP30 n.d. 1 n.d. 1 1 1 1 TP31 1 1 n.d. 1 0 0 1 TP32 n.d. 0 n.d. 1 1 02 TP33 1 1 1 0 1 2 1 TP34 n.d. n.d. n.d. n.d. 0 2 0 TP35 n.d. n.d. n.d.n.d. n.d. 1 1 TP36 0 n.d. n.d. n.d. 0 2 1 TP37 n.d. n.d. n.d. n.d. 0n.d. n.d.

The plantlets in the non-sprayed containers corresponding to constructshaving a score of 2 were transplanted at approximately 7 weeks posttransformation and grown as R0 plants using standard methods known inthe art. Plantlets corresponding to non-tolerant scores of 0 and 1 werealso grown to serve as negative controls. The R0 plants were grown in agreenhouse under long-day nursery conditions (18 hr light at 80° F. then6 hr dark at 74° F.) for approximately four weeks. At eleven weeks, theR0 plants were sprayed with two passes of the same herbicide solution(0.3 g of S-3100) described above. Herbicide injury ratings werecollected seven days after treatment.

The results of the herbicide tolerance application at eleven weeks tothe R0 plants confirmed the low percent injury rating scores observed inthe high-throughput screen at five weeks. Any injury rating of 30% orabove was equivalent to non-transgenic soybean injury ratings. A few ofthe constructs stood out as providing very good tolerance to theherbicide application. For example, TP1 with PPO H_N90 (SEQ ID NO:11)had only 3% injury and with PPO H_N30 (SEQ ID NO:14) or PPO H_N40 (SEQID NO:15) had only 5% injury; TP20 with PPO H_N90 (SEQ ID NO:11) hadonly 5% injury. In contrast, TP32 with the PPO H_N90 (SEQ ID NO:11) hadan injury score of 50%. Data are provided in Table 10, where n.d.indicates the analysis was not conducted.

TABLE 10 R0 transgenic soybean herbicide tolerance: percent injuryscores Targeting Peptide H_N20 H_N30 H_N40 H_N50 H_N90 H_N100 TP1 n.d. 5  5 n.d.  3 15 TP2 n.d. n.d. n.d. n.d. 35 n.d. TP3 n.d. n.d. n.d. n.d.30 n.d. TP4 n.d. n.d. 15 n.d. n.d. n.d. TP5 n.d. n.d. n.d. n.d. n.d.n.d. TP6 25 n.d. n.d. 40 25 30 TP7 20 n.d. 40 n.d. 15 30 TP8 n.d. n.d.30 n.d. 40 n.d. TP9 n.d. 35 n.d. 40 30 n.d. TP10 n.d. n.d. n.d. n.d. 3035 TP11 n.d. 35 n.d. n.d. 30 n.d. TP12 20 n.d. n.d. n.d. 30 50 TP13 20n.d. 15 40 15 n.d. TP14 n.d. n.d. 35 40 25 n.d. TP15 30 35 30 40 35 30TP16 n.d. n.d. n.d. n.d. 35 n.d. TP17 25 n.d. n.d. 40 15 n.d. TP18 n.d.n.d. n.d. n.d. 30 n.d. TP19 n.d. n.d. n.d. n.d. n.d. n.d. TP20 20 n.d.n.d. n.d.  5 35 TP21 n.d. n.d. 35 n.d. 25 n.d. TP22 n.d. n.d. n.d. 40 3030 TP23 n.d. n.d. n.d. n.d. 30 35 TP24 n.d. n.d. n.d. n.d. n.d. n.d.TP25 n.d. n.d. 15 35 n.d. n.d. TP26 n.d. 35 n.d. n.d. 40 n.d. TP27 n.d.n.d. n.d. n.d. 35 n.d. TP28 n.d. n.d. n.d. n.d. 30 35 TP29 30 n.d. n.d.n.d. n.d. 40 TP30 n.d. n.d. n.d. n.d. n.d. n.d. TP31 n.d. n.d. n.d. n.d.n.d. n.d. TP32 n.d. n.d. n.d. n.d. 50 40 TP33 n.d. n.d. n.d. n.d. 25n.d. TP34 n.d. n.d. n.d. n.d. 35 n.d. TP35 n.d. n.d. n.d. n.d. 15 35TP36 n.d. n.d. n.d. n.d. 15 n.d. TP37 n.d. n.d. n.d. n.d. n.d. n.d.

To further evaluate the plant transformation constructs in soybean,excised embryos (A3555) are transformed using plant transformationvectors with A. tumefaciens and standard methods known in the art.Regenerated R0 transgenic plantlets are grown in the green house andsplit into groups. The groups are sprayed with one or more PPOherbicides per group to evaluate herbicide tolerance. For example, theR0 transgenic plants are sprayed at approximately V2-V4 growth stagewith lactofen at a rate of 110 g ai/ha (0.09 lb ai/acre) or 220 g ai/ha(0.19 lb ai/acre). Plants are evaluated for injury one to fourteen daysafter treatment and injury scores are recorded. Leaf samples are used toidentify transgenic plants with a single copy of the transgenic DNAinsert, and R0 plants that contain only a single copy and pass herbicidespray testing are selfed to produce R1 seed.

R1 plants are grown in the green house and split into groups. The groupsare sprayed with one or more PPO herbicides per group to evaluateherbicide tolerance. For example, the PPO herbicide lactofen is appliedpre-emergent and/or at the V2 to V6 growth stage at a rate of 220 gai/ha (0.19 lb ai/acre). Plants are evaluated for injury one to fourteendays after treatment and injury scores are recorded. Unsprayedtransgenic plants are used for phenotypic comparison with unsprayedwild-type plants.

R2 plants are generated by selfing a homozygous transgenic R1 plant andcollecting seed. R2 plants are evaluated at one or more field locationsor greenhouse assays. Herbicide treatments are applied and plots orplants are rated for crop injury one to fourteen days after herbicideapplication on a scale of 0-100 with zero being no injury and 100 beingcomplete crop death.

Example 7 Leaf Disc Assay

A leaf-disc assay was employed for rapid and minimally damagingassessment of herbicide tolerance in transgenic plants expressing arecombinant PPO enzyme. Leaf tissue was sampled from the youngestfully-green leaf of corn and soybean plants. Five 4-mm diameter leafdiscs were cut from the leaf samples using a disposable biopsy punchwith a plunger (Integra® Miltex®, Inc., York, Pa.) and the leaf discswere placed into one ml of incubation solution (1 mM MES, pH 6.5, 1% w/vsucrose, 1% (v/v) acetone) in 24-well polystyrene plates with lids. Forherbicide tolerance analysis, S-3100 was added to the incubationsolution to a final concentration of 1 micromolar. Vacuum infiltrationwas applied and plates of leaf discs were incubated in continuousdarkness for one day at room temperature (23-24° C.) followed by one(soybean) or two (corn) days continuous light incubation under overheadfluorescent and incandescent bulbs (520 uE) at 26-27° C. Leaf discinjury was then scored visually on a scale from 0 (lowest injury) to 4(highest injury), with lower scores indicating better tolerance to thePPO herbicide.

Leaf discs from transgenic maize plants produced using construct 6 andexpressing PPO enzyme H_N10 (SEQ ID NO: 4) showed significant herbicidetolerance based upon an average leaf disc score of 0.4. This result wasconsistent with the PPO herbicide tolerance observed in whole plantstransformed with construct 6 and expressing PPO enzyme H_N10 (SEQ ID NO:4). Leaf discs from transgenic soy plants produced using construct 1 andconstruct 11 and expressing PPO enzyme H_N10 (SEQ ID NO: 4) showed thatplants with construct 1 had significant herbicide tolerance with aninjury rating of zero, while plants with construct 11 had an injuryrating of 1.6 and non-transgenic plants had an injury rating of 2.6.

Example 8 Expression and Testing of HemG PPO Enzymes in Cotton

The microbial HemG PPO enzymes are expressed in transgenic cottonplants, and the transgenic plants are analyzed for PPO herbicidetolerance. In cotton, excised embryos (Coker 130) are transformed withthese vectors using A. tumefaciens and standard methods known in theart. Regenerated R0 transgenic plantlets are grown in the green houseand split into groups. The groups are used to spray PPO herbicides (onePPO herbicide per group) to evaluate tolerance. For example, the R0transgenic plantlets are sprayed at approximately 2-4 true leaf growthstage with lactofen at a rate of 110 g ai/ha (0.09 lb ai/acre) or 220 gai/ha (0.19 lb ai/acre). Plants are evaluated for injury one to fourteendays after treatment and injury scores are recorded. Leaf samples areused to identify transgenic plants with a single copy of the transgenicinsert. R0 plants that contain only a single copy and pass herbicidespray testing are selfed to produce R1 seed.

R1 plants are grown in the green house and split into groups. The groupsare sprayed with one or more PPO herbicides per group to evaluateherbicide tolerance. For example, the PPO herbicide lactofen is appliedpre-emergent and/or at the two true leaf to first flower stage at a rateof 220 g ai/ha (0.19 lb ai/acre) (1×). Plants are evaluated for injuryone to fourteen days after treatment and injury scores are recorded.Unsprayed transgenic plants are used for phenotypic comparison withunsprayed wild-type plants.

R2 plants are generated by selfing a homozygous transgenic R1 plant andcollecting seed. R2 plants are evaluated at one or more field locationsor greenhouse assays. Herbicide treatments are applied and plots orplants are rated for crop injury one to fourteen days after herbicideapplication on a scale of 0-100 with zero being no injury and 100 beingcomplete crop death.

What is claimed is:
 1. A recombinant DNA molecule comprising aheterologous promoter operably linked to a nucleic acid sequenceencoding a protein that has at least 85% sequence identity to apolypeptide sequence selected from the group consisting of: SEQ IDNOs:1-20, wherein the protein has herbicide-insensitiveprotoporphyrinogen oxidase activity.
 2. The recombinant DNA molecule ofclaim 1, wherein the nucleic acid sequence is selected from the groupconsisting of SEQ ID NOs:22-63.
 3. The recombinant DNA molecule of claim1, wherein the protein comprises an amino acid sequence selected fromthe group consisting of SEQ ID NOs:1-20.
 4. The recombinant DNA moleculeof claim 1, wherein the heterologous promoter is functional in a plantcell.
 5. The recombinant DNA molecule of claim 4, wherein the nucleicacid sequence is operably linked to a DNA molecule encoding a targetingsequence that functions to localize an operably linked protein within acell.
 6. A DNA construct comprising the recombinant DNA molecule ofclaim
 1. 7. The DNA construct of claim 6, wherein the recombinant DNAcomprises an operably linked DNA molecule encoding a targeting sequencethat functions to localize the protein within a cell.
 8. The DNAconstruct of claim 7, wherein the protein confers herbicide tolerance tosaid cell.
 9. The DNA construct of claim 6, wherein the DNA construct ispresent in the genome of a transgenic plant, seed, or cell.
 10. Arecombinant polypeptide that comprises at least 85% sequence identity tothe full length of an amino acid sequence chosen from SEQ ID NOs:1-20,wherein the polypeptide has herbicide-insensitive protoporphyrinogenoxidase activity.
 11. A transgenic plant, seed, cell, or plant partcomprising the recombinant DNA molecule of claim
 1. 12. The transgenicplant, seed, cell, or plant part of claim 11, wherein the transgenicplant, seed, cell, or plant part comprises an additional transgenicherbicide tolerance trait.
 13. The transgenic plant, seed, cell, orplant part of claim 11, defined as comprising herbicide tolerance to atleast one PPO herbicide.
 14. A seed according to claim
 11. 15. Atransgenic plant, seed, cell, or plant part comprising the recombinantpolypeptide of claim
 10. 16. A method for conferring herbicide toleranceto a plant, seed, cell, or plant part comprising: heterologouslyexpressing in said plant, seed, cell, or plant part the recombinantpolypeptide of claim
 10. 17. The method of claim 16, wherein said plant,seed, cell, or plant part comprises protoporphyrinogen oxidase activityconferred by the recombinant polypeptide.
 18. The method of claim 16,wherein the herbicide tolerance is to at least one PPO herbicideselected from the group consisting of: acifluorfen, fomesafen, lactofen,fluoroglycofen-ethyl, oxyfluorfen, flumioxazin, azafenidin,carfentrazone-ethyl, sulfentrazone, fluthiacet-methyl, oxadiargyl,oxadiazon, pyraflufen-ethyl, saflufenacil and S-3100.
 19. A method ofplant transformation, comprising the steps of: a) introducing therecombinant DNA molecule of claim 1 into a plant cell; and b)regenerating a plant therefrom that comprises the recombinant DNAmolecule.
 20. The method of claim 19, further comprising the step ofselecting a plant that is tolerant to at least one PPO herbicide. 21.The method of claim 19, further comprising the step of crossing theregenerated plant with itself or with a second plant and collecting seedfrom the cross.
 22. A method for controlling weeds in a plant growtharea, comprising contacting a plant growth area comprising thetransgenic plant or seed of claim 11 with at least one PPO herbicide,wherein the transgenic plant or seed is tolerant to the PPO herbicideand wherein weeds are controlled in the plant growth area.
 23. A methodof identifying a nucleotide sequence encoding a protein havingprotoporphyrinogen oxidase activity, the method comprising: a)transforming an E. coli strain having a gene knockout for the native E.coli PPO enzyme with a bacterial expression vector comprising arecombinant DNA molecule encoding a candidate herbicide toleranceprotein; and b) growing said transformed E. coli using a heme-freebacterial medium, wherein growth using said bacterial medium identifiesa protein having protoporphyrinogen oxidase activity.
 24. A method ofidentifying a nucleotide sequence encoding a protein havingherbicide-insensitive protoporphyrinogen oxidase activity, the methodcomprising: a) transforming an E. coli strain having a gene knockout forthe native E. coli PPO enzyme with a bacterial expression vectorcomprising a recombinant DNA molecule encoding a recombinant protein;and b) growing said transformed E. coli using a bacterial mediumcontaining at least one PPO herbicide, wherein growth of bacteriaidentifies a protein having herbicide-insensitive protoporphyrinogenoxidase activity.
 25. A method of screening for a herbicide tolerancegene comprising: a) expressing the recombinant DNA molecule of claim 1in a plant cell; and b) identifying a plant cell that displays toleranceto a PPO herbicide.
 26. A method of screening for a herbicide tolerancegene comprising: a) expressing a recombinant DNA molecule of claim 1 ina bacterial cell lacking HemG, wherein the bacterial cell is grown in aheme-free medium in the presence of a PPO herbicide; and b) identifyinga bacterial cell that displays tolerance to a PPO herbicide.
 27. Amethod of producing a plant tolerant to a PPO herbicide and at least oneother herbicide comprising: a) obtaining a plant according to claim 11;and b) crossing the transgenic plant with a second plant comprisingtolerance to the at least one other herbicide, and c) selecting aprogeny plant resulting from said crossing that comprises tolerance to aPPO herbicide and the at least one other herbicide.
 28. A method forreducing the development of herbicide tolerant weeds comprising: a)cultivating in a crop growing environment a plant according to claim 12;and b) applying a PPO herbicide and at least one other herbicide to thecrop growing environment, wherein the crop plant is tolerant to the PPOherbicide and the at least one other herbicide.
 29. The method of claim28, wherein the PPO herbicide is selected from the group consisting ofacifluorfen, fomesafen, lactofen, fluoroglycofen-ethyl, oxyfluorfen,flumioxazin, azafenidin, carfentrazone-ethyl, sulfentrazone,fluthiacet-methyl, oxadiargyl, oxadiazon, pyraflufen-ethyl, saflufenaciland S-3100.
 30. The method of claim 28, wherein the at least one otherherbicide is selected from the group consisting of: an ACCase inhibitor,an ALS inhibitor, an EPSPS inhibitor, a synthetic auxin, aphotosynthesis inhibitor, a glutamine synthesis inhibitor, a HPPDinhibitor, a PPO inhibitor, and a long-chain fatty acid inhibitor. 31.The method of claim 30, wherein the ACCase inhibitor is anaryloxyphenoxy propionate or a cyclohexanedione; the ALS inhibitor is asulfonylurea, imidazolinone, triazoloyrimidine, or a triazolinone; theEPSPS inhibitor is glyphosate; the synthetic auxin is a phenoxyherbicide, a benzoic acid, a carboxylic acid, or a semicarbazone; thephotosynthesis inhibitor is a triazine, a triazinone, a nitrile, abenzothiadiazole, or a urea; the glutamine synthesis inhibitor isglufosinate; the HPPD inhibitor is an isoxazole, a pyrazolone, or atriketone; the PPO inhibitor is a diphenylether, a N-phenylphthalimide,an aryl triazinone, or a pyrimidinedione; or the long-chain fatty acidinhibitor is a chloroacetamide, an oxyacetamide, or a pyrazole.